Structural Information about the trans-to-cis Isomerization Mechanism of the Photoswitchable Fluorescent Protein rsEGFP2 Revealed by Multiscale Infrared Transient Absorption.

RsEGFP2 is a reversibly photoswitchable fluorescent protein used in super-resolved optical microscopies, which can be toggled between a fluorescent On state and a nonfluorescent Off state. Previous time-resolved ultraviolet-visible spectroscopic studies have shown that the Off-to-On photoactivation extends over the femto- to millisecond time scale and involves two picosecond lifetime excited states and four ground state intermediates, reflecting a trans-to-cis excited state isomerization, a millisecond deprotonation, and protein structural reorganizations. Femto- to millisecond time-resolved multiple-probe infrared spectroscopy (TRMPS-IR) can reveal structural aspects of intermediate species. Here we apply TRMPS-IR to rsEGFP2 and implement a Savitzky-Golay derivative analysis to correct for baseline drift. The results reveal that a subpicosecond twisted excited state precursor controls the trans-to-cis isomerization and the chromophore reaches its final position in the protein pocket within 100 ps. A new step with a time constant of 42 ns is reported and assigned to structural relaxation of the protein that occurs prior to the deprotonation of the chromophore on the millisecond time scale.

Enhanced Ultraviolet Photoresponse Characteristics of Indium Gallium Zinc Oxide Photo-Thin-Film Transistors Enabled by Surface Functionalization of Biomaterials for Real-Time Ultraviolet Monitoring.

Indium gallium zinc oxide (IGZO) is one of the most promising materials for diverse optoelectronic applications based on thin-film transistors (TFTs) including ultraviolet (UV) photodetectors. In particular, the monitoring of UV-A (320-400 nm) exposure is very useful for healthcare applications because it can be used to prevent various human skin and eye-related diseases. However, the relatively weak optical absorption in the UV-A range and the persistent photoconductivity (PPC) arising from the oxygen vacancy-related states of IGZO thin films limit efficient UV monitoring. In this paper, we report the enhancement of the UV photoresponse characteristics of IGZO photo-TFTs by the surface functionalization of biomolecules on an IGZO channel. The biomaterial/IGZO interface plays a crucial role in enhancing UV-A absorption and suppressing PPC under negative gate bias, resulting in not only increased photoresponsivity and specific detectivity but also a fast and repeatable UV photoresponse. In addition, turning off the device without external bias completely eliminates PPC due to the internal electric field induced by the surface functionalization of biomaterials. Such a volatile feature of PPC enables the fast and repeatable UV photoresponse. These results suggest the potential of IGZO photo-TFTs combined with biomaterials for real-time UV monitoring.

Erasable labeling of neuronal activity using a reversible calcium marker.

Understanding how the brain encodes and processes information requires the recording of neural activity that underlies different behaviors. Recent efforts in fluorescent protein engineering have succeeded in developing powerful tools for visualizing neural activity, in general by coupling neural activity to different properties of a fluorescent protein scaffold. Here, we take advantage of a previously unexploited class of reversibly switchable fluorescent proteins to engineer a new type of calcium sensor. We introduce rsCaMPARI, a genetically encoded calcium marker engineered from a reversibly switchable fluorescent protein that enables spatiotemporally precise marking, erasing, and remarking of active neuron populations under brief, user-defined time windows of light exposure. rsCaMPARI photoswitching kinetics are modulated by calcium concentration when illuminating with blue light, and the fluorescence can be reset with violet light. We demonstrate the utility of rsCaMPARI for marking and remarking active neuron populations in freely swimming zebrafish.

Electrostatic control of photoisomerization pathways in proteins.

Rotation around a specific bond after photoexcitation is central to vision and emerging opportunities in optogenetics, super-resolution microscopy, and photoactive molecular devices. Competing roles for steric and electrostatic effects that govern bond-specific photoisomerization have been widely discussed, the latter originating from chromophore charge transfer upon excitation. We systematically altered the electrostatic properties of the green fluorescent protein chromophore in a photoswitchable variant, Dronpa2, using amber suppression to introduce electron-donating and electron-withdrawing groups to the phenolate ring. Through analysis of the absorption (color), fluorescence quantum yield, and energy barriers to ground- and excited-state isomerization, we evaluate the contributions of sterics and electrostatics quantitatively and demonstrate how electrostatic effects bias the pathway of chromophore photoisomerization, leading to a generalized framework to guide protein design.

OLIVe: A Genetically Encoded Fluorescent Biosensor for Quantitative Imaging of Branched-Chain Amino Acid Levels inside Single Living Cells.

Branched-chain amino acids (BCAAs) are essential amino acids, controlling cellular metabolic processes as signaling molecules; therefore, utilization of intracellular BCAAs may be regulated by the availability of nutrients in the environment. However, spatial and temporal regulation of intracellular BCAA concentration in response to environmental conditions has been unclear due to the lack of suitable methods for measuring BCAA concentrations inside single living cells. Here, we developed a Förster resonance energy transfer (FRET)-based genetically encoded biosensor for BCAAs, termed optical biosensor for leucine-isoleucine-valine (OLIVe). The biosensor showed approximately 2-fold changes in FRET values corresponding to BCAA concentrations. Importantly, FRET signals from HeLa cells expressing OLIVe in the cytoplasm and nucleus correlated with bulk intracellular BCAA concentrations determined from populations of cells by a biochemical method, and were decreased by knockdown of L-type amino acid transporter 1 (LAT1), a transporter for BCAAs, indicating that OLIVe can reliably report intracellular BCAA concentrations inside single living cells. We also succeeded in imaging BCAA concentrations in the mitochondria using mitochondria-targeted OLIVe. Using the BCAA imaging technique, we found apparently correlated concentrations between the cytoplasm and the mitochondria. We also found that extracellular non-BCAA amino acids affected intracellular BCAA concentrations. Of these amino acids, extracellular glutamine markedly increased intracellular BCAA concentrations in a LAT1-dependent manner. Unexpectedly, extracellular pyruvate was also found to have significant positive effects on maintaining intracellular BCAA concentrations, suggesting that the cells have pyruvate-dependent systems to import BCAAs and/or to regulate BCAA metabolism.

Fluorescent Protein-Based Indicators for Functional Super-Resolution Imaging of Biomolecular Activities in Living Cells.

Super-resolution light microscopy (SRM) offers a unique opportunity for diffraction-unlimited imaging of biomolecular activities in living cells. To realize such potential, genetically encoded indicators were developed recently from fluorescent proteins (FPs) that exhibit phototransformation behaviors including photoactivation, photoconversion, and photoswitching, etc. Super-resolution observations of biomolecule interactions and biochemical activities have been demonstrated by exploiting the principles of bimolecular fluorescence complementation (BiFC), points accumulation for imaging nanoscale topography (PAINT), and fluorescence fluctuation increase by contact (FLINC), etc. To improve functional nanoscopy with the technology of genetically encoded indicators, it is essential to fully decipher the photo-induced chemistry of FPs and opt for innovative indicator designs that utilize not only fluorescence intensity but also multi-parametric readouts such as phototransformation kinetics. In parallel, technical improvements to both the microscopy optics and image analysis pipeline are promising avenues to increase the sensitivity and versatility of functional SRM.

Photocleavable Cadherin Inhibits Cell-to-Cell Mechanotransduction by Light.

Precise integration of individual cell behaviors is indispensable for collective tissue morphogenesis and maintenance of tissue integrity. Organized multicellular behavior is achieved via mechanical coupling of individual cellular contractility, mediated by cell adhesion molecules at the cell-cell interface. Conventionally, gene depletion or laser microsurgery has been used for functional analysis of intercellular mechanotransduction. Nevertheless, these methods are insufficient to investigate either the spatiotemporal dynamics or the biomolecular contribution in cell-cell mechanical coupling within collective multicellular behaviors. Herein, we present our effort in adaption of PhoCl for attenuation of cell-to-cell tension transmission mediated by E-cadherin. To release intercellular contractile tension applied on E-cadherin molecules with external light, a genetically encoded photocleavable module called PhoCl was inserted into the intracellular domain of E-cadherin, thereby creating photocleavable cadherin (PC-cadherin). In response to light illumination, the PC-cadherin cleaved into two fragments inside cells, resulting in attenuating mechanotransduction at intercellular junctions in living epithelial cells. Light-induced perturbation of the intercellular tension balance with surrounding cells changed the cell shape in an epithelial cell sheet. The method is expected to enable optical manipulation of force-mediated cell-to-cell communications in various multicellular behaviors, which contributes to a deeper understanding of embryogenesis and oncogenesis.

Spontaneously Blinking Fluorescent Protein for Simple Single Laser Super-Resolution Live Cell Imaging.

Super-resolution imaging techniques based on single molecule localization microscopy (SMLM) broke the diffraction limit of optical microscopy in living samples with the aid of photoswitchable fluorescent probes and intricate microscopy systems. Here, we developed a fluorescent protein, SPOON, which can be switched off by excitation light illumination and switched on by thermally induced dehydration, resulting in an apparent spontaneous blinking behavior. This unique property of SPOON provides a simple SMLM-based super-resolution imaging platform which requires only a single 488 nm laser.

Chimeric Autofluorescent Proteins as Photophysical Model System for Multicolor Bimolecular Fluorescence Complementation.

The yellow fluorescent protein (YFP) is frequently used in a protein complementation assay called bimolecular fluorescence complementation (BiFC), and is employed to visualize protein-protein interactions. In this analysis, two different, nonfluorescent fragments of YFP are genetically attached to proteins of interest. Upon interaction of these proteins, the YFP fragments are brought into proximity close enough to reconstitute their original structure, enabling fluorescence. BiFC allows for a straightforward readout of protein-protein interactions and furthermore facilitates their functional investigation by in vivo imaging. Furthermore, it has been observed that the available color range in BiFC can be extended upon complementing fragments of different proteins that are, like YFP, derived from the Aequorea victoria green fluorescent protein, thereby allowing for a multiplexed investigation of protein-protein interactions. Some spectral characteristics of "multicolor" BiFC (mcBiFC) complexes have been reported before; however, no in-depth analysis has been performed yet. Therefore, little is known about the photophysical characteristics of these mcBiFC complexes because a proper characterization essentially relies on in vitro data. This is particularly difficult for fragments of autofluorescent proteins (AFPs) because they show a very strong tendency to form supramolecular aggregates which precipitate ex vivo. In this study, this intrinsic difficulty is overcome by directly fusing the coding DNA of different AFP fragments. Translation of the genetic sequence in Escherichia coli leads to fully functional, highly soluble fluorescent proteins with distinct properties. On the basis of their construction, they are designated chimeric AFPs, or BiFC chimeras, here. Comparison of their spectral characteristics with experimental in vivo BiFC data confirmed the utility of the chimeric proteins as a BiFC model system. In this study, nine different chimeras were thoroughly analyzed at both the ensemble and the single-molecular level. The data indicates that mutations believed to be photophysically silent significantly alter the properties of AFPs.

A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein.

Far-red fluorescent proteins (FPs) are desirable for in vivo imaging because with these molecules less light is scattered, absorbed, or re-emitted by endogenous biomolecules compared with cyan, green, yellow, and orange FPs. We developed a new class of FP from an allophycocyanin α-subunit (APCα). Native APC requires a lyase to incorporate phycocyanobilin. The evolved FP, which we named small ultra-red FP (smURFP), covalently attaches a biliverdin (BV) chromophore without a lyase, and has 642/670-nm excitation-emission peaks, a large extinction coefficient (180,000 M(-1)cm(-1)) and quantum yield (18%), and photostability comparable to that of eGFP. smURFP has significantly greater BV incorporation rate and protein stability than the bacteriophytochrome (BPH) FPs. Moreover, BV supply is limited by membrane permeability, and smURFPs (but not BPH FPs) can incorporate a more membrane-permeant BV analog, making smURFP fluorescence comparable to that of FPs from jellyfish or coral. A far-red and near-infrared fluorescent cell cycle indicator was created with smURFP and a BPH FP.

In vivo near-infrared fluorescence imaging of Leishmania amazonensis expressing infrared fluorescence protein (iRFP) for real-time monitoring of cutaneous leishmaniasis in mice.

The use of Leishmania amazonensis-infected BALB/c mice is an important model for the study of experimental cutaneous leishmaniasis. Here we report the development of a non-invasive method to directly evaluate and measure parasite burden during the course of the infection, based on the near-infrared fluorescence detection of a recombinant L. amazonensis strain. So, we generated a L. amazonensis strain that stably expresses the near-infrared protein (iRFP) gene and compared the maintenance of its vitro and in vivo characteristics, such as fitness, pathogenicity and fluorescence emission. After that, we followed the disease development, as well as the parasite burden in BALB/c mice footpads infected with L. amazonensis-iRFP, by using an in vivo near-infrared fluorescence scanner. In vitro results showed a linear correlation between the fluorescence emission and the number of parasites. The in vivo study showed that the use of iRFP-transfected L. amazonensis enables the monitoring of parasite burden by measuring fluorescence signals. Therefore, this technique can be confidently used to directly monitor parasitic load and infection overtime and could be an excellent tool for in vitro and in vivo screening of anti-leishmanial drugs and vaccine efficiency. This is the first report of the use of the near-infrared fluorescence imaging technique for monitoring in vivo cutaneous leishmaniasis.

Optical biosensors.

Optical biosensors represent the most common type of biosensor. Here we provide a brief classification, a description of underlying principles of operation and their bioanalytical applications. The main focus is placed on the most widely used optical biosensors which are surface plasmon resonance (SPR)-based biosensors including SPR imaging and localized SPR. In addition, other optical biosensor systems are described, such as evanescent wave fluorescence and bioluminescent optical fibre biosensors, as well as interferometric, ellipsometric and reflectometric interference spectroscopy and surface-enhanced Raman scattering biosensors. The optical biosensors discussed here allow the sensitive and selective detection of a wide range of analytes including viruses, toxins, drugs, antibodies, tumour biomarkers and tumour cells.

The crystal structure of red fluorescent protein TagRFP-T reveals the mechanism of its superior photostability.

The red fluorescent protein variant TagRFP-T has greatly improved photostability over its parent molecule, TagRFP, but the underlying mechanism leading to this improvement is to date unknown. The 1.95 Å resolution crystallographic structure of TagRFP-T showed that its chromophore exists as a mixture of cis and trans coplanar isomers in roughly equal proportions. Interestingly, both isomers are able to fluoresce, a property that has never been observed in any other fluorescent protein. We propose a "circular restoration model" for TagRFP-T to explain its superior photostability: There are four co-existing chromophore states (cis/trans protonated/ionized state) that can be driven by light to transform from one state into another. This model also explains how TagRPF-T essentially eliminates the temporary dark state (reversible photobleaching).

Photo-convertible fluorescent proteins as tools for fresh insights on subcellular interactions in plants.

Optical highlighters comprise photo-activatable, photo-switchable and photo-convertible fluorescent proteins and are relatively recent additions to the toolbox utilized for live cell imaging research. Here, we provide an overview of four photo-convertible fluorescent proteins (pcFP) that are being used in plant cell research: Eos, Kaede, Maple and Dendra2. Each of these proteins has a significant advantage over other optical highlighters since their green fluorescent nonconverted forms and red fluorescent converted forms are generally clearly visible at expression levels that do not appear to interfere with subcellular dynamics and plant development. These proteins have become increasingly useful for understanding the role of transient and sustained interactions between similar organelles. Tracking of single organelles after green-to-red conversion has provided novel insights on plastids and their stroma-filled extensions and on the formation of mega-mitochondria. Similarly colour recovery after photo-conversion has permitted the estimation of nuclear endo-reduplication events and is being developed further to image protein trafficking within the lumen of the endoplasmic reticulum. We have also applied photo-convertible proteins to create colour-differentiation between similar cell types to follow their development. Both the green and red fluorescent forms of these proteins are compatible with other commonly used single coloured FPs. This has allowed us to develop simultaneous visualization schemes for up to five types of organelles and investigate organelle interactivity. The advantages and caveats associated with the use of photo-convertible fluorescent proteins are discussed.

KillerOrange, a Genetically Encoded Photosensitizer Activated by Blue and Green Light.

Genetically encoded photosensitizers, proteins that produce reactive oxygen species when illuminated with visible light, are increasingly used as optogenetic tools. Their applications range from ablation of specific cell populations to precise optical inactivation of cellular proteins. Here, we report an orange mutant of red fluorescent protein KillerRed that becomes toxic when illuminated with blue or green light. This new protein, KillerOrange, carries a tryptophan-based chromophore that is novel for photosensitizers. We show that KillerOrange can be used simultaneously and independently from KillerRed in both bacterial and mammalian cells offering chromatic orthogonality for light-activated toxicity.

Optical control of biological processes by light-switchable proteins.

Cellular processes such as proliferation, differentiation, or migration depend on precise spatiotemporal coordination of protein activities. Correspondingly, reaching a quantitative understanding of cellular behavior requires experimental approaches that enable spatial and temporal modulation of protein activity. Recently, a variety of light-sensitive protein domains have been engineered as optogenetic actuators to spatiotemporally control protein activity. In the present review, we discuss the principle of these optical control methods and examples of their applications in modulating signaling pathways. By controlling protein activity with spatiotemporal specificity, tunable dynamics, and quantitative control, light-controllable proteins promise to accelerate our understanding of cellular and organismal biology.

Pulse-shaping based two-photon FRET stoichiometry.

Förster Resonance Energy Transfer (FRET) based measurements that calculate the stoichiometry of intermolecular interactions in living cells have recently been demonstrated, where the technique utilizes selective one-photon excitation of donor and acceptor fluorophores to isolate the pure FRET signal. Here, we present work towards extending this FRET stoichiometry method to employ two-photon excitation using a pulse-shaping methodology. In pulse-shaping, frequency-dependent phases are applied to a broadband femtosecond laser pulse to tailor the two-photon excitation conditions to preferentially excite donor and acceptor fluorophores. We have also generalized the existing stoichiometry theory to account for additional cross-talk terms that are non-vanishing under two-photon excitation conditions. Using the generalized theory we demonstrate two-photon FRET stoichiometry in live COS-7 cells expressing fluorescent proteins mAmetrine as the donor and tdTomato as the acceptor.

Quantitative single cell gene expression profiling in the avian embryo.

BACKGROUND: Single cell gene profiling has been successfully applied to cultured cells. However, isolation and preservation of a cell's native gene expression state from an intact embryo remain problematic. RESULTS: Here, we present a strategy for in vivo single cell profiling that optimizes cell identification, isolation and amplification of nucleic acids with nominal bias and sufficient material detection. We first tested several photoconvertible fluorescent proteins to selectively mark a cell(s) of interest in living chick embryos then accurately identify and isolate the same cell(s) in fixed tissue slices. We determined that the dual color mDendra2 provided the optimal signal/noise ratio for this purpose. We developed proper procedures to minimize cell death and preserve gene expression, and suggest nucleic acid amplification strategies for downstream analysis by microfluidic reverse transcriptase quantitative polymerase chain reaction or RNAseq. Lastly, we compared methods for single cell isolation and found that our fluorescence-activated cell sorting (FACS) protocol was able to preserve native transcripts and generate expression profiles with much higher efficiency than laser capture microdissection (LCM). CONCLUSIONS: Quantitative single cell gene expression profiling may be accurately applied to interrogate complex cell dynamics events during embryonic development by combining photoconversion cell labeling, FACS, proper handling of isolated cells, and amplification strategies.

Microfluidics-based selection of red-fluorescent proteins with decreased rates of photobleaching.

Fluorescent proteins offer exceptional labeling specificity in living cells and organisms. Unfortunately, their photophysical properties remain far from ideal for long-term imaging of low-abundance cellular constituents, in large part because of their poor photostability. Despite widespread engineering efforts, improving the photostability of fluorescent proteins remains challenging due to lack of appropriate high-throughput selection methods. Here, we use molecular dynamics guided mutagenesis in conjunction with a recently developed microfluidic-based platform, which sorts cells based on their fluorescence photostability, to identify red fluorescent proteins with decreased photobleaching from a HeLa cell-based library. The identified mutant, named Kriek, has 2.5- and 4-fold higher photostability than its progenitor, mCherry, under widefield and confocal illumination, respectively. Furthermore, the results provide insight into mechanisms for enhancing photostability and their connections with other photophysical processes, thereby providing direction for ongoing development of fluorescent proteins with improved single-molecule and low-copy imaging capabilities.

In cellulo evaluation of phototransformation quantum yields in fluorescent proteins used as markers for single-molecule localization microscopy.

Single-molecule localization microscopy of biological samples requires a precise knowledge of the employed fluorescent labels. Photoactivation, photoblinking and photobleaching of phototransformable fluorescent proteins influence the data acquisition and data processing strategies to be used in (Fluorescence) Photoactivation Localization Microscopy ((F)-PALM), notably for reliable molecular counting. As these parameters might depend on the local environment, they should be measured in cellulo in biologically relevant experimental conditions. Here, we measured phototransformation quantum yields for Dendra2 fused to actin in fixed mammalian cells in typical (F)-PALM experiments. To this aim, we developed a data processing strategy based on the clustering optimization procedure proposed by Lee et al (PNAS 109, 17436-17441, 2012). Using simulations, we estimated the range of experimental parameters (molecular density, molecular orientation, background level, laser power, frametime) adequate for an accurate determination of the phototransformation yields. Under illumination at 561 nm in PBS buffer at pH 7.4, the photobleaching yield of Dendra2 fused to actin was measured to be (2.5 ± 0.4) × 10(-5), whereas the blinking-off yield and thermally-activated blinking-on rate were measured to be (2.3 ± 0.2) × 10(-5) and 11.7 ± 0.5 s-1, respectively. These phototransformation yields differed from those measured in poly-vinyl alcohol (PVA) and were strongly affected by addition of the antifading agent 1,4-diazabicyclo[2.2.2]octane (DABCO). In the presence of DABCO, the photobleaching yield was reduced 2-fold, the blinking-off yield was decreased more than 3-fold, and the blinking-on rate was increased 2-fold. Therefore, DABCO largely improved Dendra2 photostability in fixed mammalian cells. These findings are consistent with redox-based bleaching and blinking mechanisms under (F)-PALM experimental conditions. Finally, the green-to-red photoconversion quantum yield of Dendra2 was estimated to be (1.4 ± 0.6) × 10(-5) in cellulo under 405 nm illumination.